The present invention concerns a method for the detection of cytosine methylations in DNA. 5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis (for review: Millar et al.: Five not four: History and significance of the fifth base. In: S. Beck and A. Olek, eds.: The Epigenome. Wiley-VCH Verlag Weinheim, 2003, p. 3-20). The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. 5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behaviour as cytosine. In addition, in the case of a PCR amplification, the epigenetic information, which is borne by 5-methylcytosines, is completely lost.
The usual methods for methylation analysis operate essentially according to two different principles. Either methylation-specific restriction enzymes are utilized, or a selective chemical conversion of unmethylated cytosines to uracil is conducted (bisulfite treatment). The enzymatically or chemically pretreated DNA is then amplified and can be analyzed in different ways (for review: Fraga and Esteller: DNA Methylation: A Profile of Methods and Applications. Biotechniques 33: 632-649, September 2002; WO 02/072880, pp. 1 ff).
As the use of methylation-specific enzymes is restricted to certain sequences containing restriction sites recognised by said enzymes, for most applications a bisulfite treatment is conducted (for review: U.S. Ser. No. 10/311,661).
According to the invention a “bisulfite reaction”, “bisulfite treatment” or “bisulfite method” shall mean a reaction for the conversion of cytosine bases in a nucleic acid to uracil bases in the presence of bisulfite ions whereby 5-methyl-cytosine bases are not significantly converted. The bisulfite reaction contains a deamination step and a desulfonation step which can be conducted separately or simultaneously (further details are described and a reaction scheme is shown in EP 1394172 A1, incorporated by reference herein in its entirety). There are various documents addressing specific aspects of the bisulfite reaction, including Hayatsu et al., Biochemistry 9 (1970) 2858-28659; Slae and Shapiro, J. Org. Chem. 43 (1978) 4197-4200; Paulin et al., Nucl. Acids Res. 26 (1998) 5009-5010; Raizis et al., Anal. Biochem. 226 (1995), 161-1666; Wang et al. Nucleic Acids Res. 8 (1980) 4777-4790. These documents are summarized in EP 1394172 A1 (incorporated by reference herein in its entirety).
The bisulfite treatment is usually conducted in the following way: The genomic DNA is isolated, mechanically or enzymatically fragmentised, denaturated by NaOH, converted several hours by a concentrated bisulfite solution and finally desulfonated and desalted (e.g.: Frommer et al.: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl. Acad. Sci. USA 1992 Mar. 1; 89(5):1827-31; incorporated by reference herein in its entirety).
In recent times several technical improvements of the bisulfite methods were developed. The agarose bead method incorporates the DNA to be investigated in an agarose matrix, through which diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek A. et al. A modified and improved method for bisulphite based cytosine methylation analysis, Nucl. Acids Res. 1996, 24, 5064-5066). In the patent application WO 01/98528 (=DE 100 29 915; =U.S. application Ser. No. 10/311,661) a bisulfite conversion is described in which the DNA sample is incubated with a bisulfite solution of a concentration range between 0.1 mol/l to 6 mol/l in presence of a denaturating reagent and/or solvent and at least one scavenger. In said patent application several suitable denaturating reagents and scavengers are described (document incorporated by reference herein in its entirety). In the patent application WO 03/038121 (=DE 101 54 317; =U.S. Ser. No. 10/416,624) a method is disclosed in which the DNA to be analysed is bound to a solid surface during the bisulfite treatment. Consequently, purification and washing steps are facilitated. Further improvements are described in the patent applications EP 1394173 A1 and EP 1394172 A1 (incorporated by reference herein in its entirety). The patent application PCT/EP2004/011715 (=DE 103 47 396.3; DE 103 47 397.1; DE 103 47 400.5; DE 103 47 399.8) describes an improved bisulfite treatment by using dioxane or n-alkylene glycol compounds in combination with a special temperature program and a special purification step by ultrafiltration. In said patent application a reaction temperature of 50° C. for 5 h is described (Example 2).
However, a basic problem of the bisulfite treatment consists of the fact that long reaction times are necessary in order to assure a complete conversion and to exclude false-positive results. Simultaneously, however, this leads to a degradation of the DNA due to the long reaction times. Higher reaction temperatures in fact lead to a higher conversion rate, but also to a more intense degradation of the DNA. The interactions between temperature, reaction time, rates of conversion and degradation were recently investigated systematically. In this way, it could be shown that the highest conversion rates were attained at temperatures of 55° C. (with reaction times between 4 and 18 hours) and at 95° C. (with a reaction time of one hour). A serious problem, however, is the degradation of the DNA during this procedure. It is described that at a reaction temperature of 55° C., 84-96% of DNA is decomposed. At 95° C. the degradation is even higher (Grunau et al.: Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 2001 Jul. 1; 29(13):E65-5; incorporated by reference herein in its entirety). Thus, most authors use reaction temperatures of approximately 50° C. (see: Frommer et al., loc. cit. 1992, p. 1827; Olek et al., loc. cit. 1996, p. 5065; Raizis et al: A bisulfite method of 5-methylcytosine mapping that minimizes template degradation. Anal Biochem. 1995 Mar. 20; 226(1):161-6, 162). In the patent application WO 2004/067545 A1 (applicant: Hoffmann-La Roche) an improved bisulfite treatment is disclosed using a reaction temperature between 70 and 90° C. for 1.5 to 3.5 hours.
Due to the high losses of the conventional bisulfite treatment, it is problematic to use these methods for investigations in which the quantity of DNA to be analyzed is limited. A particularly interesting field of methylation analysis, however, lies in diagnosing cancer diseases or other disorders associated with a change in methylation status by means of analysis of DNA from bodily fluids, e.g. from blood or urine. However, DNA is present only in small concentrations in body fluids, so that the applicability of methylation analysis is limited by the low yield of conventional bisulfite treatment.
Accordingly, based on the particular importance of cytosine methylation analysis and based on the described disadvantages of conventional methodology, there is a great technical need for improved methods of bisulfite conversion.
It was now found that under certain optimized reaction conditions using denaturating reagents, a reaction temperature >50° C., particularly >55° C., and a reaction time ≧5 h the conversion rate of the bisulfite reaction is increased in an unexpected, surprising way. This could be shown by a new method enabling an exact determination of the conversion rate of the DNA (see EP 05 075 404.3, incorporated by reference in its entirety). In a preferred embodiment of the invention certain time-temperature combinations are used (60° C./5 h and 55° C./7 h). Usually the bisulfite conversion using denaturating reagents is conducted at 50° C. for 5 h (see example 2 in PCT/EP2004/011715).
Although it is known to the person skilled in the art that increases in temperature or time can increase the performance of reaction, the person skilled in the art could not expect that the herein disclosed time-temperature rise would cause such a strong effect, particularly because it was thought that an time/temperature increase would lead to a higher degradation of the DNA (see above). The new method, however, leads to a higher amount of converted, aplificable DNA. The new method is particularly applicable for the analysis of DNA derived from bodily fluids, e.g. blood or plasma. In combination with additives, improved reaction conditions and/or new purification methods the efficacy of the conversion can be further improved. Thereby a sensitive DNA methylation analysis of bodily fluids becomes possible.
A further aspect of the present invention relates to the use of particular radical scavengers in the bisulfite reaction.
The use of radical scavengers for the bisulfite reaction is already known (DE 10029915), but most of the known methods use hydroquinone as a radical scavenger (see: Fromer et al: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA, 1992 Mar. 1; 89 (5):1827-31).